Abstract Three dimensional genome organization is emerging as a potent regulator of gene expression. Due to recent advancements in methods used to study higher-order chromatin structure, there has been a dramatic increase in the understanding of how the human genome is organized in three dimensional space. This has led to a greater appreciation for the role of 3D genome architecture in regulating nuclear biology, and a recognition of the role of alterations to normal 3D genome structure in causing human disease. Despite this, the mechanisms used by cells to build higher-order chromatin structures remain relatively obscure. The experiments in this proposal will shed light on the mechanisms of higher-order chromatin structure formation using two primary approaches. First, how cells construct their own 3D genome architectures will be studied by taking advantage of naturally occurring biological systems where chromatin structures are formed and re- formed. This will be accomplished by studying changes that occur during re-formation of chromatin structure after exit from mitosis, and by studying the formation of new chromatin loops in the genome upon change in cellular identity. Second, to determine how higher-order chromatin structures are built, CRISPR/Cas9 technology will be used to engineer exogenous chromatin loops into the genome. This will be accomplished by delivering protein cargo using Cas9 fusion proteins to specific loci in the genome to perform locus specific ?re- programming.? This will be used to assess what are the minimal essential events that are required to create higher-order chromatin structures in the genome. These experiments will greatly expand our knowledge of the mechanisms used to construct higher-order chromatin structures in our genome and will provide the necessary foundation to link our knowledge of 3D genome organization to human phenotypic variation and disease.